A new nomogram model for prognosis of hepatocellular carcinoma based on novel gene signature that regulates cross-talk between immune and tumor cells.

BACKGROUND: The combined application of immune cells and specific biomarkers related to the tumor immune microenvironment has a better predictive value for the prognosis of HCC. The purpose of this study is to construct a new prognostic model based on immune-related genes that regulate cross-talk between immune and tumor cells to assess the prognosis and explore possible mechanisms. METHOD: The immune cell abundance ratio of 424 cases in the TCGA-LIHC database is obtained through the CIBERSORT algorithm. The differential gene analysis and cox regression analysis is used to screen IRGs. In addition, the function of IRGs was preliminarily explored through the co-culture of M2 macrophages and HCC cell lines. The clinical validation, nomogram establishment and performing tumor microenvironment score were validated. RESULTS: We identified 4 immune cells and 9 hub genes related to the prognosis. Further, we identified S100A9, CD79B, TNFRSF11B as an IRGs signature, which is verified in the ICGC and GSE76427 database. Importantly, IRGs signature is closely related to the prognosis, tumor microenvironment score, clinical characteristics and immunotherapy, and nomogram combined with clinical characteristics is more conducive to clinical promotion. In addition, after co-culture with M2 macrophages, the migration capacity and cell pseudopod of MHCC97H increased significantly. And CD79B and TNFRSF11B were significantly down-regulated in MHCC97H, Huh7 and LM3, while S100A9 was up-regulated. CONCLUSION: We constructed an IRGs signature and discussed possible mechanisms. The nomogram established based on IRGs can accurately predict the prognosis of HCC patients. These findings may provide a suitable therapeutic target for HCC.

Long noncoding RNA HULC regulates the NF-κB pathway and represents a promising prognostic biomarker in liver cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) are involved in a diverse array of biological processes. While lncRNAs are commonly upregulated in hepatocellular carcinoma (HCC), the specific regulatory roles they play in this oncogenic context require further study and clarification. Although HULC (lncRNA highly upregulated in liver cancer) is involved in disease pathogenesis, its precise role in this context remains unclear. METHODS: Here, we have explored the mechanistic relevance of HULC expression by assessing its expression in patient samples. The importance of this lncRNA in the onset and progression of HCC was investigated through in vitro approaches including Western blotting, quantitative PCR, Transwell assays, electron microscopy, wound healing assays, and real-time cell analysis (RTCA). Additionally, the in vivo functions of this lncRNA were assessed using an orthotopic HCC xenograft in nude mouse model system. RESULTS: HULC was identified as a lncRNA that is highly upregulated in human liver tumors. In vitro, HULC was able to promote HCC malignancy, although its excess overexpression also led robust autophagic induction, promoting the increased expression of autophagy-associated genes including LC3 and Beclin-1. At a mechanistic level, HULC was able to promote the phosphorylation of p65 and IkBkB thus enhancing autophagy by increasing LC3II levels in a manner dependent upon the NF-κB pathway. HULC downregulation was also linked to impaired orthotopic HCC tumor growth in vivo. The link between HULC and autophagy may play a role in disease progression. CONCLUSIONS: These results suggest that HULC is an oncogenic lncRNA, and may thus offer value as a prognostic biomarker and promoter of HCC development, in addition to being a potential therapeutic target in this cancer type.

NIR-II imaging of hepatocellular carcinoma based on a humanized anti-GPC3 antibody.

Liver cancer, of which hepatocellular carcinoma (HCC) is the most common form, is one of the most lethal cancers worldwide. The five-year survival rate for HCC is below 9%, which can be attributed to late diagnosis and limited treatment options at the late stage. Therefore, safe and efficient imaging strategies are urgently needed to facilitate HCC diagnosis and stage evaluation. The development of the second near infrared window (NIR-II, 1000-1700 nm) fluorescence imaging offers the advantages of enhanced resolutions, deeper penetration depth, and less autofluorescence compared to traditional NIR-I window (700-900 nm) imaging. Herein, an HCC targeted NIR-II fluorescent probe, GPC-ICG, was developed by labelling a humanized anti-GPC3 monoclonal antibody with indocyanine green (ICG). Compared to the negative control IgG-ICG probe, the GPC3-ICG probe demonstrated specific GPC3 targeting capability in vitro. And for GPC3 positive Huh-7 tumor bearing mice, the GPC3-ICG probe specifically accumulated in subcutaneous xenografts, with a tumor-background ratio (TBR) of up to 3. The NIR-II imaging of mice organs ex vivo also indicated that GPC3-ICG specifically targeted Huh-7 tumor tissue. Overall, GPC3-ICG is a promising NIR-II probe for GPC3 targeted imaging of HCC.

RIPK4 Suppresses the Invasion and Metastasis of Hepatocellular Carcinoma by Inhibiting the Phosphorylation of STAT3.

Receptor interacting serine/threonine kinase 4 (RIPK4) is a member of the threonine/serine protein kinase family; it plays related functions in a variety of tumours, but its biological function has not been fully revealed. It has been reported that it is differentially expressed in hepatocellular carcinoma (HCC). Our research aimed to reveal the role of RIPK4 in the progression of HCC and to reveal the biological behaviour of RIPK4 in HCC. We analysed the differences in RIPK4 expression in HCC by using a publicly available data set. By using PCR, Western blotting and immunohistochemical staining methods, we detected the expression level of RIPK4 in HCC patient specimens and studied the relationship between the expression of RIPK4 and the clinicopathological features of HCC patients. The prognostic data were combined to analyse the relationship between RIPK4 and HCC patient survival and tumour recurrence. We found that the expression level of RIPK4 in nontumour tissues was significantly higher than that in tumour tissues, and the level of RIPK4 was significantly positively correlated with postoperative survival and recurrence in HCC patients. Further, our study found that RIPK4 inhibits the progression of HCC by influencing the invasion and metastasis of HCC and that overexpression of RIPK4 reduces the invasion and metastasis of HCC by inhibiting epithelial-mesenchymal transition (EMT) and the STAT3 pathway. In in vivo experiments, overexpression of RIPK4 stably inhibited HCC metastasis. To summarize, our research revealed the relationship between RIPK4 and the prognosis of patients with HCC. We discovered that RIPK4 affects the invasion and metastasis of HCC through the EMT and STAT3 pathways. Targeted inhibition of the RIPK4 gene and the STAT3 pathway may be potential therapeutic strategies for inhibiting the postoperative recurrence and metastasis of HCC.

A Humanized Anti-GPC3 Antibody for Immuno-Positron Emission Tomography Imaging of Orthotopic Mouse Model of Patient-Derived Hepatocellular Carcinoma Xenografts.

Glypican-3 (GPC3) is an attractive diagnostic marker for hepatocellular carcinoma (HCC). We previously reported the potential of an 89Zr-labeled murine anti-GPC3 antibody (clone 1G12) for immunoPET imaging of HCC in orthotopic patient-derived xenograft (PDX) mouse models. We now humanized the murine antibody by complementarity determining region (CDR) grafting, to allow its clinical translation for human use. The engineered humanized anti-GPC3 antibody, clone H3K3, retained comparable binding affinity and specificity to human GPC3. H3K3 was conjugated with desferrioxamine (Df) and radiolabeled with 89Zr to produce the PET/CT tracer 89Zr-Df-H3K3. When injected into GPC3-expressing orthotopic HCC PDX in NOD SCID Gamma (NSG) mice, 89Zr-Df-H3K3 showed specific high uptake into the orthotopic PDX and minimal, non-specific uptake into the non-tumor bearing liver. Specificity was demonstrated by significantly higher uptake of 89Zr-Df-H3K3 into the non-blocked PDX mice, compared with the blocked PDX mice (which received prior injection of 100 mg of unlabeled H3K3). Region of interest (ROI) analysis showed that the PDX/non-tumor liver ratio was highest (mean ± SD: 3.4 ± 0.31) at 168 h post injection; this ratio was consistent with biodistribution studies at the same time point. Thus, our humanized anti-GPC3 antibody, H3K3, shows encouraging potential for use as an immunoPET tracer for diagnostic imaging of HCC patients.

Alternative Splicing-Based Differences Between Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma: Genes, Immune Microenvironment, and Survival Prognosis.

Alternative splicing (AS) event is a novel biomarker of tumor tumorigenesis and progression. However, the comprehensive analysis of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) is lacking. Differentially expressed analysis was used to identify the differentially expressed alternative splicing (DEAS) events between HCC or ICC tissues and their normal tissues. The correlation between DEAS events and functional analyses or immune features was evaluated. The cluster analysis based on DEAS can accurately reflect the differences in the immune microenvironment between HCC and ICC. Forty-five immune checkpoints and 23 immune features were considered statistically significant in HCC, while only seven immune checkpoints and one immune feature in ICC. Then, the prognostic value of DEAS events was studied, and two transcripts with different basic cell functions (proliferation, cell cycle, invasion, and migration) were produced by ADHFE1 through alternative splicing. Moreover, four nomograms were established in conjunction with relevant clinicopathological factors. Finally, we found two most significant splicing factors and further showed their protein crystal structure. The joint analysis of the AS events in HCC and ICC revealed novel insights into immune features and clinical prognosis, which might provide positive implications in HCC and ICC treatment.

An NIR-II/MR dual modal nanoprobe for liver cancer imaging.

Hepatocellular carcinoma (HCC) is a malignancy of the liver worldwide and surgical resection remains the most effective treatment. However, it is still a great challenge to locate small lesions and define the border of diffused HCC even with the help of preoperative imaging examination. Here, we reported a rare-earth-doped nanoparticle NaGdF4:Nd 5%@NaGdF4@Lips (named Gd-REs@Lips), which simultaneously performed powerful functions in both magnetic resonance imaging (MRI) and second near-infrared fluorescence window imaging (NIR-II, 1000-1700 nm). Imaging studies on orthotopic models with xenografts established from HCC patients indicated that Gd-REs@Lips efficiently worked as a T2-weighted imaging contrast agent to increase the signal intensity difference between liver cancer tissues and surrounding normal liver tissues on MRI, and it can also serve as a negative NIR-II imaging contrast enabling the precise detection of liver cancer. More importantly, benefiting from the high sensitivity of NIR-II imaging, Gd-REs@Lips allowed the visualization of tiny metastasis lesions (2 mm) on the liver surface. It is expected that the dual NIR-II/MRI modal nanoprobe developed holds high potential to fill the gap between the preoperative imaging detection of cancer lesions and intra-operative guidance, and it further brings new opportunities to address HCC-related medical challenges.